ABSTRACT Cellular differentiation, development and homeostasis depend on regulation of gene expression, which is largely focused on the DNA transcription initiation process. During transcription initiation, Mediator, a large multi-protein complex conserved throughout eukaryotes, conveys regulatory signals to RNA polymerase II (RNAPII), the enzyme responsible for transcription of all protein-coding genes. Depending on the specific organism, Mediator can include 25-29 different polypeptides (total MW 1-1.5MDa) organized into Head, Middle, Tail and CDK8 Kinase (CKM) modules, but its enzymatic activity is limited to a single Cdk8 kinase subunit. Mounting evidence points to a mechanism largely based on conformational rearrangements that modulate Mediator interaction with RNAPII. Consequently, a detailed understanding of Mediator structure and its conformational dynamics is essential to elucidate how the complex regulates initiation. Macromolecular electron microscopy (cryo-EM) is the technique of choice for characterization of large, dynamic macromolecular assemblies. In the last couple of years, cryo-EM studies of Mediator have dramatically advanced our molecular understanding of the complex. Here we propose cryo-EM, biochemical, and functional studies of yeast and mammalian Mediators that build on our previous work, and that will reveal in molecular detail the way in which critical factors modulate Mediator conformation and interaction with RNAPII, bringing about regulation of transcription initiation. In Aim 1 We will calculate cryo EM maps of mouse (MmMED) and human (HsMED) Mediators at near-atomic resolution, localize metazoan-specific subunits, and determine how these subunits affect Mediator structural rearrangements and RNAPII interaction. These results will provide an atomic-resolution understanding of mammalian Mediator structure and reveal the structural basis for specific details of transcription regulation by mammalian Mediator. In Aim 2 we will use cryo-EM and biochemistry to determine the effect of CKM binding on Mediator conformation and RNAPII association, and will investigate regulation of CKM interaction with Mediator. This will lead to an understanding of how Mediator interaction with the CKM and concomitant structural changes influence Mediator association with RNAPII, holoenzyme formation and, ultimately, transcription initiation. In Aim 3 we will use cryo-EM, image analysis and biochemistry to understand how binding of activators and repressors to yeast and mammalian Mediators influences Mediator conformation, interaction with RNAPII and gene expression. These studies will reveal how interaction with activators and repressors, which generally target subunits (mostly in the Tail module) not directly involved in RNAPII interaction, can ultimately have an effect on regulation of transcription initiation by Mediator. Results from the studies we propose will provide a detailed understanding of mammalian Mediator structure, and reveal how conformational regulation and interactions enable transcription regulation by Mediator in all eukaryotes. Parallel analysis of yeast and mammalian Mediators will reveal fundamental aspects of the regulation mechanism related to structural conservation of Mediator across eukaryotes, while highlighting aspects of regulation specific to the more intricate mammalian Mediator. The work we propose continues our strategy of combining structural analysis with biochemical and functional studies, and will depend critically on 1) application of state-of-the-art cryo-EM, in which my group has considerable expertise; 2) strong, ongoing collaborations with research groups that are leaders in biochemical and functional studies of mammalian Mediator.